Mouse anti βiii tubulin

Cells were fixed with 4% paraformaldehyde. Cell-specific proteins were identified by indirect immunofluorescence using mouse anti-β-III-tubulin (1:2000; R&D Systems); rabbit anti-GFAP (1:500; Dako) and rabbit anti-MBP (1:250; Dako) antisera. The anti-rabbit DyLight 568- and anti-mouse AlexaFluor 488-labeled (1:500; Invitrogen) were used as secondary antisera. For nuclear staining, cells were incubated with 1 μg/mL Hoechst 33258.

To detect proteins via immunofluorescence, cells were grown on glass coverslips, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked in a mixture of 10% NGS (Jackson ImmunoResearch, Cambridge, UK) and 5% BSA (Sigma) before incubation with a primary antibody. The following primary antibodies were used: mouse anti-nestin (1:500, Millipore, Burlington, MA, USA), mouse anti-SOX2 (1:500, Sigma), rabbit anti-Ki-67 (1:400, DBS, Pleasanton, CA, USA), mouse anti-phospho histone H2A.X (γH2AX, 1:300, Millipore) and mouse anti-β III-tubulin (1:1000, R&D Systems). Nestin, SOX2, γH2AX and β III-tubulin staining was visualized by goat anti-mouse IgG Alexa Fluor 488 secondary antibody (Invitrogen); Ki-67 staining was visualized by goat anti-rabbit IgG Alexa Fluor 488 secondary antibody (Invitrogen). To visualize nuclei, cells were stained with DAPI (Sigma). Representative pictures were acquired using a Leica DM6000 microscope. The percentage of Ki-67-positive cells, number of γH2AX foci per nuclei, and percentage of βIII-tubulin-positive cells were counted manually using the ImageJ software (version 1.48, NIH, USA; Cell Counter plugin). Multiple fields (between 300–500 cells) per each staining in four independent experiments were counted.